Changes in synaptic connections are considered essential for learning and memory formation1–6. However, it is unknown how neural circuits undergo continuous synaptic changes during learning while maintaining lifelong memories. Here we show, by following postsynaptic dendritic spines over time in the mouse cortex7–8, that learning and novel sensory experience lead to spine formation and elimination by a protracted process. The extent of spine remodelling correlates with behavioural improvement after learning, suggesting a crucial role of synaptic structural plasticity in memory formation and storage. Importantly, a small fraction of new spines induced by novel experience, together with most spines formed early during development and surviving experience-dependent elimination, are preserved throughout the entire life of an animal. These studies indicate that learning and daily sensory experience leave minute but permanent marks on cortical connections and suggest that lifelong memories are stored in largely stably connected synaptic networks.

Determining the degree of synapse formation and elimination is essential for understanding the structural basis of brain plasticity and pathology. We show that in vivo imaging of dendritic spine dynamics through an open-skull glass window, but not a thinned-skull window, is associated with high spine turnover and substantial glial activation during the first month after surgery. These findings help to explain existing discrepancies in the degree of dendritic spine plasticity observed in the mature cortex.

Imaging neurons, glia and vasculature in the living brain has become an important experimental tool for understanding how the brain works. Here we describe in detail a protocol for imaging cortical structures at high optical resolution through a thinned-skull cranial window in live mice using two-photon laser scanning microscopy (TPLSM). Surgery can be performed within 30–45 min and images can be acquired immediately thereafter. The procedure can be repeated multiple times allowing longitudinal imaging of the cortex over intervals ranging from days to years. Imaging through a thinned-skull cranial window avoids exposure of the meninges and the cortex, thus providing a minimally invasive approach for studying structural and functional changes of cells under normal and pathological conditions in the living brain.

Fragile X syndrome (FXS) is the most common inherited form of mental retardation and is caused by transcriptional inactivation of the X-linked fragile X mental retardation 1 (FMR1) gene. FXS is associated with increased density and abnormal morphology of dendritic spines, the postsynaptic sites of the majority of excitatory synapses. To better understand how lack of the FMR1 gene function affects spine development and plasticity, we examined spine formation and elimination of layer 5 pyramidal neurons in the whisker barrel cortex of Fmr1 KO mice with a transcranial two-photon imaging technique. We found that the rates of spine formation and elimination over days to weeks were significantly higher in both young and adult KO mice compared with littermate controls. The heightened spine turnover in KO mice was due to the existence of a larger pool of "short-lived" new spines in KO mice than in controls. Furthermore, we found that the formation of new spines and the elimination of existing ones were less sensitive to modulation by sensory experience in KO mice. These results indicate that the loss of Fmr1 gene function leads to ongoing overproduction of transient spines in the primary somatosensory cortex. The insensitivity of spine formation and elimination to sensory alterations in Fmr1 KO mice suggest that the developing synaptic circuits may not be properly tuned by sensory stimuli in FXS.autism | imaging | mental retardation | synaptic plasticity | two-photon microscopy F ragile X syndrome (FXS) is the most common form of inherited mental retardation, affecting about 1 in 4,000 males and 1 in 8,000 females (1). Patients who suffer from FXS exhibit various degrees of cognitive, socio-affective, and sensory-motor abnormalities (2). The syndrome is caused by the expansion of a polymorphic CGG trinucleotide repeat in the 5′ untranslated region of the fragile X mental retardation 1 (FMR1) gene located on the X chromosome (3). The fragile X mental retardation protein (FMRP), which is encoded by the FMR1 gene, binds to many mRNAs and is believed to regulate protein translation in various subcellular locations, including dendrites and dendritic spines (4, 5).The Fmr1 KO mice demonstrate many abnormalities found in FXS patients, such as impairments of learning and memory (6-8), social behaviors (9-11), and sensory processing (12, 13), thus providing an excellent model system to study pathogenic mechanisms underlying FXS. Despite these behavioral abnormalities, the gross structure of the brain is largely intact in FXS patients and in the mouse model of the disorder. The most consistent anatomical finding is an abnormal profile of dendritic spines, postsynaptic protrusions that receive the vast majority of excitatory input in the brains of diverse species (14-17).In FXS, the adult dendritic spine phenotype includes increases in spine density and spine length and the number of immaturelooking spines in the various brain regions examined (15,16,18). Similarly, in the visual and somatosensory cortices of adult Fmr1 KO mice, p...

Key Points Deletion/haploinsufficiency of Asxl1 causes MDS-like disease in mice. Asxl1 loss reduces the HSC pool and decreases HSC hematopoietic repopulating capacity in vivo.